This project has examined two aspects of T lymphocyte programmed cell death: (1)The roles of calpain and reactive oxygen intermediates in the TcR-triggered events in the "activation-induced" death of mature T cells, which involves TcR-induced Fas ligand upregulation and subsequent Fas crosslinking; (2) the role of ICE-family proteases (caspases) as common downstream mediators of multiple pathways of apoptotic death in all stages of T lymphocyte development. Previous results had shown that the TcR-induced death pathway in hybridomas and blasts is blocked by both protease inhibitors and antioxidants, and we have continued to define the mechanisms of these inhibitions. Specific inhibitors of the calcium-dependent cysteine protease calpain selectively blocked TcR induced FasL mRNA upregulation. These inhibitors blocked activation-induced FasL promoter activity as assessed by transfection with a luciferase reporter construct. Antioxidants behave like calpain inhibitors in that most block the TcR-induced Fas ligand upregulation at the mRNA transcription level. Activation-induced reactive oxygen intermediates were detected in hybridomas by dihydrorhodamine oxidation as detected by flow cytometry. We have addressed the role of ICE-family proteases (caspases) as downstream mediators of apoptotic death in T lymphocytes by testing the ability of caspase inhibitors to block death in various T cells induced by different stimuli. We have principally used the peptide-fluoromethyl ketone caspase inhibitors ZVAD-FMK (an inhibitor of ICE and CPP32) and BD-FMK (an inhibitor of CPP32 but not ICE). Both compounds completely and specifically block all readouts of thymocyte death by four independent pathways. When resting peripheral T cells were examined, ZVAD-FMK was somewhat less effective than BD-FMK, while with T cell blasts, ZVAD-FMK was considerably less potent an inhibitor than BD-FMK. The apoptotic death of CTLL-2 cells was inhibited by BD-FMK but not by ZVAD-FMK. These results argue that caspases do play a critical functional role in all T cell apoptotic death, but the differential sensitivity to these inhibitors suggests that different caspase family members are critical in different T cells and stimuli.